Figure 5. The effects of biliatresone are secondary to decreased GSH.

(a) GSH levels were measured in cholangiocytes in 2D culture over 24 h following treatment with biliatresone. Mean ± SEM, *, p<0.05 (compared to 0 time point). (b) Cholangiocyte spheroids in 3D culture were treated with the compounds indicated for 24 h and were then immunostained for F-actin (green) and with the nuclear stain DAPI (blue). Only spheroids that were fully visualized along the z axis were scored. (c) Spheroids treated as in a, immunostained for cellular tubulin (red). d) Quantification of the spheroids from a. Spheroids (96–119) in three different experiments for each condition were graded as open (lumen wide open), partially closed (small lumen), or closed (obstructed lumen). e) Immunofluorescence staining of neonatal mouse extrahepatic bile ducts incubated for 24 h in high oxygen environment (see methods), treated with the compounds indicated and immunostained for K19 (green) and α-SMA (red). Size bars for a,b: 10 μM; for e: 100 μM. Representative images from (a,b), 3 independent experiments, each experiment done in duplicate. (e) from 4 independent experiments with at least 8 ducts total for each condition.